Neutral tumor evolution in myeloma is associated with poor prognosis

Recent studies suggest that the evolutionary history of a cancer is important in forecasting clinical outlook. To gain insight into the clonal dynamics of multiple myeloma (MM) and its possible influence on patient outcome we analysed whole exome sequencing tumor data for 333 patients from Myeloma XI, a UK phase III trial and 434 patients from the CoMMpass study, all of which had received immunomodulatory therapy (IMiD). By analysing mutant allele frequency distributions in tumors we found that 17-20% of MM is under neutral evolutionary dynamics. These tumors are associated with poorer patient survival in non-intensively treated patients, consistent with reduced therapeutic efficacy of micro-environment modulating IMiD drugs. Our findings provide evidence that knowledge of the evolutionary history of MM has relevance for predicting patient outcome and personalising therapy

Study of mechanisms controlling the efficacy and the specificity of GnRH receptor signaling : identification and role of the partner protein SET

La fonction de reproduction est sous le contrôle de la neurohormone hypothalamique GnRH qui régule la synthèse et la libération des gonadotropines hypophysaires. La GnRH agit par l’intermédiaire d’un récepteur couplé aux protéines G exprimé à la surface des cellules gonadotropes, le récepteur de la GnRH (RGnRH). Ce récepteur, chez les mammifères, a la particularité d’être dépourvu de queue C terminale ce qui le rend insensible aux systèmes classiques de désensibilisation. Ainsi, les mécanismes qui régulent l’efficacité et la spécificité de sa signalisation demeurent mal connus. Nous avons recherché des partenaires d’interaction du RGnRH, jusqu’alors inconnus, avec l’idée que ces protéines en interagissant avec les domaines intracellulaires du récepteur influenceraient son couplage aux voies de signalisation. Nos travaux ont permis d’identifier le premier partenaire d’interaction du RGnRH : la protéine SET. Par des expériences de « GST pull down », nous avons montré que SET interagit directement avec le RGnRH via le premier domaine intracellulaire du récepteur. Cette interaction implique des séquences riches en acides aminés basiques sur le récepteur et les domaines N- et C-terminaux de SET. Nous avons également montré, par co-immunoprécipitation, que le RGnRH dans sa conformation native interagit avec la protéine SET dans les cellules gonadotropes alphaT3-1 et, par immunocytochimie, que les deux protéines colocalisent à la membrane plasmique. En développant au laboratoire des outils biosenseurs permettant de mesurer avec une grande sensibilité et en temps réel les variations intracellulaires de calcium et d’AMPc, nous avons mis en évidence que le RGnRH se couple non seulement à la voie calcique mais aussi à la voie AMPc dans la lignée alphaT3-1, apportant pour l’AMPc la première démonstration d’un tel couplage. En utilisant différentes stratégies expérimentales visant à diminuer ou au contraire favoriser l’interaction du récepteur avec SET (ARN antisens, peptide correspondant à la première boucle intracellulaire du récepteur, surexpression de SET), nous avons montré que SET induit une réorientation de la signalisation du RGnRH de la voie calcique vers la voie AMPc. Nos résultats concernant l’activité du promoteur du gène du Rgnrh nous conduisent à postuler que SET pourrait favoriser l’induction par la GnRH de gènes régulés via la voie AMPc et notamment celui codant le RGnRH. Nos travaux mettent également en évidence que la GnRH régule non seulement l’expression de la protéine SET dans les cellules gonadotropes mais aussi son degré de phosphorylation favorisant ainsi sa relocalisation dans le cytoplasme des cellules alphaT3-1. Ceci suggère que la GnRH exerce une boucle de régulation permettant d’amplifier l’action de SET sur la signalisation de son propre récepteur. Enfin, nous avons mis en évidence que l’expression de SET est fortement augmentée dans l’hypophyse au moment du prœstrus chez le rat, apportant ainsi la première démonstration d’une variation de SET dans un contexte physiologique. Étant donné que le couplage du RGnRH à la voie de signalisation AMPc est augmenté au moment du prœstrus, nos résultats suggèrent que SET pourrait jouer un rôle important in vivo en favorisant ce couplage à ce stade particulier du cycle œstrien.Reproductive function is under the control of the hypothalamic neurohormone GnRH, which regulates the synthesis and the release of pituitary gonadotropins. GnRH acts on a G-protein coupled receptor expressed at the surface of pituitary gonadotrope cells, the GnRH receptor (GnRHR). This receptor, in mammals, is unique because it is devoided of the C terminal tail, which makes it insensitive to classical desensitization processes. Therefore, the mechanisms that regulate the efficacy and the specificity of its signaling are still poorly known. We searched for interacting partners of GnRHR with the idea that these proteins by interacting with the intracellular domains of the receptor could influence receptor coupling to its signaling pathways. Our work identified the first interacting partner of GnRHR: the protein SET. By GST pull down assays, we showed that SET interacts directly with GnRHR through the first intracellular loop of the receptor. This interaction involves sequences enriched in basic amino acids in the receptor and both N- and C terminal domains of SET. We also showed, by co-immunoprecipitation, that GnRHR in its native conformation interacts with the endogenous SET protein in gonadotrope alphaT3-1 cells and, by immunocytochemistry that the two proteins colocalize at the plasma membrane. By developing in the laboratory biosensors tools that allow to measure with high sensitivity and in real-time intracellular variations in calcium and cAMP concentrations, we demonstrated that GnRHR couples not only to the calcium pathway but also to the cAMP pathway in alphaT3-1 cell line, providing for cAMP the first demonstration of such coupling. Using several experimental strategies to reduce or increase receptor interaction with SET (small interfering RNA, peptide corresponding to the first intracellular loop of the receptor, overexpression of SET), we have shown that SET induces a switch of GnRHR signaling from calcium to cAMP pathway. Our results concerning the activity of the Gnrhr gene promoter led us to postulate that SET could favor the induction by GnRH of genes regulated through the cAMP pathway, notably those encoding the GnRHR. Our study also showed that GnRH regulates not only SET protein expression in gonadotropes, but also its phosphorylation level leading to its relocation in the cytoplasm of alphaT3-1 cells. This suggests that GnRH induces a regulatory loop to amplify SET action on signaling of its own receptor. Finally, we demonstrated that SET expression is markedly increased in the pituitary gland at prœstrus in female rats, providing the first demonstration of a variation of SET expression in a physiological context. Given that GnRHR coupling to the cAMP pathway is increased at prœstrus, our results suggest that SET may play an important role in vivo by promoting such coupling at this particular stage of the estrus cycle

Étude des mécanismes contrôlant l'efficacité et la spécificité de la signalisation du récepteur de la GnRH (identification et rôle de la protéine partenaire SET)

La fonction de reproduction est sous le contrôle de la neurohormone hypothalamique GnRH qui régule la synthèse et la libération des gonadotropines hypophysaires. La GnRH agit par l intermédiaire d un récepteur couplé aux protéines G exprimé à la surface des cellules gonadotropes, le récepteur de la GnRH (RGnRH). Ce récepteur, chez les mammifères, a la particularité d être dépourvu de queue C terminale ce qui le rend insensible aux systèmes classiques de désensibilisation. Ainsi, les mécanismes qui régulent l efficacité et la spécificité de sa signalisation demeurent mal connus. Nous avons recherché des partenaires d interaction du RGnRH, jusqu alors inconnus, avec l idée que ces protéines en interagissant avec les domaines intracellulaires du récepteur influenceraient son couplage aux voies de signalisation. Nos travaux ont permis d identifier le premier partenaire d interaction du RGnRH : la protéine SET. Par des expériences de GST pull down , nous avons montré que SET interagit directement avec le RGnRH via le premier domaine intracellulaire du récepteur. Cette interaction implique des séquences riches en acides aminés basiques sur le récepteur et les domaines N- et C-terminaux de SET. Nous avons également montré, par co-immunoprécipitation, que le RGnRH dans sa conformation native interagit avec la protéine SET dans les cellules gonadotropes alphaT3-1 et, par immunocytochimie, que les deux protéines colocalisent à la membrane plasmique. En développant au laboratoire des outils biosenseurs permettant de mesurer avec une grande sensibilité et en temps réel les variations intracellulaires de calcium et d AMPc, nous avons mis en évidence que le RGnRH se couple non seulement à la voie calcique mais aussi à la voie AMPc dans la lignée alphaT3-1, apportant pour l AMPc la première démonstration d un tel couplage. En utilisant différentes stratégies expérimentales visant à diminuer ou au contraire favoriser l interaction du récepteur avec SET (ARN antisens, peptide correspondant à la première boucle intracellulaire du récepteur, surexpression de SET), nous avons montré que SET induit une réorientation de la signalisation du RGnRH de la voie calcique vers la voie AMPc. Nos résultats concernant l activité du promoteur du gène du Rgnrh nous conduisent à postuler que SET pourrait favoriser l induction par la GnRH de gènes régulés via la voie AMPc et notamment celui codant le RGnRH. Nos travaux mettent également en évidence que la GnRH régule non seulement l expression de la protéine SET dans les cellules gonadotropes mais aussi son degré de phosphorylation favorisant ainsi sa relocalisation dans le cytoplasme des cellules alphaT3-1. Ceci suggère que la GnRH exerce une boucle de régulation permettant d amplifier l action de SET sur la signalisation de son propre récepteur. Enfin, nous avons mis en évidence que l expression de SET est fortement augmentée dans l hypophyse au moment du prœstrus chez le rat, apportant ainsi la première démonstration d une variation de SET dans un contexte physiologique. Étant donné que le couplage du RGnRH à la voie de signalisation AMPc est augmenté au moment du prœstrus, nos résultats suggèrent que SET pourrait jouer un rôle important in vivo en favorisant ce couplage à ce stade particulier du cycle œstrien.Reproductive function is under the control of the hypothalamic neurohormone GnRH, which regulates the synthesis and the release of pituitary gonadotropins. GnRH acts on a G-protein coupled receptor expressed at the surface of pituitary gonadotrope cells, the GnRH receptor (GnRHR). This receptor, in mammals, is unique because it is devoided of the C terminal tail, which makes it insensitive to classical desensitization processes. Therefore, the mechanisms that regulate the efficacy and the specificity of its signaling are still poorly known. We searched for interacting partners of GnRHR with the idea that these proteins by interacting with the intracellular domains of the receptor could influence receptor coupling to its signaling pathways. Our work identified the first interacting partner of GnRHR: the protein SET. By GST pull down assays, we showed that SET interacts directly with GnRHR through the first intracellular loop of the receptor. This interaction involves sequences enriched in basic amino acids in the receptor and both N- and C terminal domains of SET. We also showed, by co-immunoprecipitation, that GnRHR in its native conformation interacts with the endogenous SET protein in gonadotrope alphaT3-1 cells and, by immunocytochemistry that the two proteins colocalize at the plasma membrane. By developing in the laboratory biosensors tools that allow to measure with high sensitivity and in real-time intracellular variations in calcium and cAMP concentrations, we demonstrated that GnRHR couples not only to the calcium pathway but also to the cAMP pathway in alphaT3-1 cell line, providing for cAMP the first demonstration of such coupling. Using several experimental strategies to reduce or increase receptor interaction with SET (small interfering RNA, peptide corresponding to the first intracellular loop of the receptor, overexpression of SET), we have shown that SET induces a switch of GnRHR signaling from calcium to cAMP pathway. Our results concerning the activity of the Gnrhr gene promoter led us to postulate that SET could favor the induction by GnRH of genes regulated through the cAMP pathway, notably those encoding the GnRHR. Our study also showed that GnRH regulates not only SET protein expression in gonadotropes, but also its phosphorylation level leading to its relocation in the cytoplasm of alphaT3-1 cells. This suggests that GnRH induces a regulatory loop to amplify SET action on signaling of its own receptor. Finally, we demonstrated that SET expression is markedly increased in the pituitary gland at prœstrus in female rats, providing the first demonstration of a variation of SET expression in a physiological context. Given that GnRHR coupling to the cAMP pathway is increased at prœstrus, our results suggest that SET may play an important role in vivo by promoting such coupling at this particular stage of the estrus cycle.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

GnRH regulates the expression of its receptor accessory protein SET in pituitary gonadotropes.

Reproductive function is under the control of the neurohormone GnRH, which activates a G-protein-coupled receptor (GnRHR) expressed in pituitary gonadotrope cells. GnRHR activates a complex signaling network to regulate synthesis and secretion of the two gonadotropin hormones, luteinizing hormone and follicle-stimulating hormone, both regulating gametogenesis and steroidogenesis in gonads. Recently, in an attempt to identify the mechanisms underlying GnRHR signaling plasticity, we identified the first interacting partner of GnRHR, the proto-oncogene SET. We showed that SET binds to intracellular domains of GnRHR to enhance its coupling to cAMP pathway in αT3-1 gonadotrope cells. Here, we demonstrate that SET protein is rapidly regulated by GnRH, which increases SET phosphorylation state and decreases dose-dependently SET protein level. Our results highlight a post-translational regulation of SET protein involving the proteasome pathway. We determined that SET phosphorylation upon GnRH stimulation is mediated by PKC and that PKC mediates GnRH-induced SET down-regulation. Phosphorylation on serine 9 targets SET for degradation into the proteasome. Furthermore, a non-phosphorylatable SET mutant on serine 9 is resistant to GnRH-induced down-regulation. Altogether, these data suggest that GnRH-induced SET phosphorylation on serine 9 mediates SET protein down-regulation through the proteasome pathway. Noteworthy, SET down-regulation was also observed in response to pulsatile GnRH stimulation in LβT2 gonadotrope cells as well as in vivo in prepubertal female mice supporting its physiological relevance. In conclusion, this study highlights a regulation of SET protein by the neurohormone GnRH and identifies some of the mechanisms involved

Common coupling map advances GPCR-G protein selectivity

Two-thirds of human hormones and one-third of clinical drugs act on membrane receptors that couple to G proteins to achieve appropriate functional responses. While G protein transducers from literature are annotated in the Guide to Pharmacology database, two recent large-scale datasets now expand the receptor-G protein ‘couplome’. However, these three datasets differ in scope and reported G protein couplings giving different coverage and conclusions on G protein-coupled receptor (GPCR)-G protein signaling. Here, we report a common coupling map uncovering novel couplings supported by both large-scale studies, the selectivity/promiscuity of GPCRs and G proteins, and how the co-coupling and co-expression of G proteins compare to the families from phylogenetic relationships. The coupling map and insights on GPCR-G protein selectivity will catalyze advances in receptor research and cellular signaling toward the exploitation of G protein signaling bias in design of safer drugs

Genomics of Multiple Myeloma

International audienceMultiple myeloma (MM) is characterized by wide variability in the chromosomal/genetic changes present in tumor plasma cells. Genetically, MM can be divided into two groups according to ploidy and hyperdiploidy versus nonhyperdiploidy. Several studies in gene expression profiling attempted to identify subentities in MM without convincing results. These studies mostly confirmed the cytogenetic data and subclassified patients according to 14q32 translocations and ploidy. More-recent data that are based on whole-exome sequencing have confirmed this heterogeneity and show many gene mutations but without a unifying mutation. These newer studies have shown the frequent alteration of the mitogen-activated protein kinase pathway. The most interesting data have demonstrated subclonality in all patients with MM, including subclonal mutations of supposed driver genes KRAS, NRAS, and BRAF

Pulsatile native GnRH induces SET protein down-regulation in LβT2 cells.

<p>(a) LβT2 gonadotrope cells were treated with GnRHa (100 nM) for 2 hours and SET protein (<i>left panel</i>) and SET mRNA (<i>right panel</i>) levels were determined by Western blotting and by real-time RT-PCR, respectively. A representative immunoblot of SET expression is shown. Results are normalized by vinculin signals (SET protein) or by mRNA levels of <i>Hprt</i> (SET mRNA) and are expressed as percentage of the amount of SET in unstimulated cells. Results are expressed as mean ± SEM from 3 independent experiments. Data were analyzed by t test for unpaired groups *: p<0.05, compared to no GnRHa. (b) LβT2 gonadotrope cells were cultured in perifusion chambers as described in “Materials and methods” and challenged or not with pulsatile GnRH (10 nM) at high and low frequencies (one pulse every 0.5 hour or one pulse every 2 hours, respectively). At the end of the incubation, proteins and mRNA were extracted and SET protein and <i>Set</i>, <i>Lhb and Fshb</i> transcripts levels were determined by Western blotting and by real-time RT-PCR, respectively. A representative immunoblot of SET expression is shown. Results are normalized by vinculin signals (SET protein) or by mRNA levels of <i>Hprt</i> (<i>Set</i>, <i>Lhb and Fshb</i> mRNA) and expressed as percentage of the amount of SET in unstimulated cells. Results are expressed as mean ± SEM from 3 independent experiments. One-way ANOVA followed by Tukey’s test, ***: p<0.001, compared to no GnRH.</p

GnRH induces SET protein down-regulation in infantile mice pituitary.

<p>(a) <i>Left panel</i>—SET protein abundance was analyzed in infantile pituitaries (7–17 dpn) by Western blotting, with GAPDH used as a loading control. Bar graphs show the mean ± SEM of SET levels normalized to those of GAPDH (n = 3 to 8 pituitaries/age). A representative immunoblot of SET expression is shown. Data were analyzed by one-way ANOVA, followed by Tukey’s test with distinct letters indicating significant differences between ages (p<0.05). <i>Right panel</i>—The relative pituitary abundance of SET mRNA in infantile (7–17 dpn) females was determined by real-time RT-PCR and normalized to the mRNA levels of <i>Hprt</i> (n = 4 to 8 pituitaries/age). Bar graphs show the mean ± SEM of relative quantification. Data were analyzed by one-way ANOVA, followed by Tukey’s test with distinct letters indicating significant differences between ages (p<0.05). (b) SET protein abundance was analyzed in infantile pituitaries after treatment with the GnRH antagonist Ganirelix or saline vehicle at 12 and 13 dpn. A representative blot is shown. Bar graphs show the mean ± SEM of SET levels normalized to those of GAPDH (n = 3 to 5 pituitaries/condition). Data were analyzed by t test for unpaired groups, **: p<0.01, compared to 14 dpn saline.</p

GnRHa increases SET phosphorylation–Involvement of PKC.

<p>(a) <i>Left panel–</i>αT3-1 gonadotrope cells were plated in 6-well plates and labelled with [³²P]-orthophosphate (50 μCi/ml) as described in “Materials and methods”. Cells were stimulated (+) or not (-) with GnRHa (100 nM) for 0.5 hour followed by SET immunoprecipitation (IP SET) as described in “Materials and methods”. Immunoprecipitated SET was resolved on 10% SDS-PAGE, electrotransferred and probed with anti-SET antibody (IB SET). Phosphorylated SET (P-SET) was visualized by autoradiography using a Fuji Phosphoimager FLA7000. <i>Right panel–</i>αT3-1 gonadotrope cells were incubated (+) or not (-) with GnRHa (100 nM, 0.5 hour) and phosphoproteins were purified by chromatography as described in “Materials and methods”. Phosphorylated SET and ERK1/2 were detected by Western blotting using anti-SET and anti-total ERK1/2 antibodies, respectively. Results are representative of 4 independent experiments. (b) αT3-1 gonadotrope cells were pre-incubated or not with the PKC inhibitor GF109203X (2 μM, 1 hour) prior to GnRHa stimulation (100 nM, 0.5 hour). Phosphoproteins were purified by chromatography as described in “Materials and methods” and phosphorylated SET was detected by Western blotting using anti-SET antibody. Results are expressed as the percentage of SET protein level in absence of treatment. Results are expressed as mean ± SEM from 3 independent experiments. Data were analyzed by Two-way ANOVA followed by Tukey’s test, with distinct letters indicating significant differences between treatments (p<0.05). (c) αT3-1 gonadotrope cells were pre-incubated or not with the PKC inhibitor GF109203X (2 μM, 1 hour) prior to GnRHa stimulation (100 nM, 0.5 hour) and SET protein expression was detected by Western blotting using anti-SET antibody. Results are normalized by vinculin signals and expressed as the percentage of SET protein level in absence of treatment. Results are expressed as mean ± SEM from 3 to 4 independent experiments. Data were analyzed by Two-way ANOVA followed by Tukey’s test, with distinct letters indicating significant differences between treatments (p<0.05).</p